Abnormal fibrinogens IJmuiden (B beta Arg14----Cys) and Nijmegen (B beta Arg44----Cys) form disulfide-linked fibrinogen-albumin complexes.

Koopman, J.; Haverkate, F.; Grimbergen, Jos; Engesser, L.; Nováková, I.R.O.; Kerst, A. F. J. A.; Lord, Susan T.

doi:10.1073/pnas.89.8.3478

Cited by 59 publications

(58 citation statements)

References 30 publications

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“…Immunoblot analysis ofplasma. To determine whether the additional cysteine created by the mutation in fibrinogen Dusart (Aa 554 Arg --Cys) is involved in disulfide-linked complex formation with other proteins (33)(34)(35), plasma ofthe propositus, his siblings, and normal plasma was analyzed by immunoblotting after separation of plasma proteins on 2-16% gradient SDS-PAGE gels. The blots were incubated with a mouse monoclonal antifibrinogen antibody (Y18) and a goat …”

Section: Resultsmentioning

confidence: 99%

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

Koopman

Haverkate²,

Grimbergen³

et al. 1993

J. Clin. Invest.

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The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C -> T) in the Aa-chain gene, resulting in the amino acid substitution Aa 554 Arg Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (Aa 554 Arg -n Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals. (J. Clin. Invest. 1993.

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Section: Resultsmentioning

confidence: 99%

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

Koopman

Haverkate²,

Grimbergen³

et al. 1993

J. Clin. Invest.

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111

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“…Because these 2 bands reacted with an antibody against human albumin, they likely represent HMW and LMW fibrinogen complexed with albumin ( Figure 3B), similar to earlier reports for the fibrinogen variants IJmuiden (B␤ R14C) and Nijmegen (B␤ R44C). 20 SDS-PAGE of reduced samples revealed that the A␣-, B␤-, and ␥-chains from normal and variant fibrinogen were identical and stained with the same intensity. No abnormally migrating chains were visible (data not shown).…”

Section: Sds-page and Immunoblot Analysis Of Fibrinogenmentioning

confidence: 99%

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

Bolliger-Stucki

Lord

Furlan

2001

Blood

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Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A␣ R16C and ␥ G165R. As seen previously with other heterozygous A␣ R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A␣ 16 C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the ␣-helix content in the variant D3 fragment. It is concluded that the A␣-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the ␥-chain induced a conformational change in the D3 fragment. IntroductionFibrinogen, a soluble plasma glycoprotein of 340 kd, is made up of 2 copies of 3 different polypeptide chains (A␣ 2 , B␤ 2 ,␥ 2 ), linked together with 29 interchain and intrachain disulfide bridges. The molecule is organized in a dimeric fashion consisting of a central E domain containing the amino termini of all 6 polypeptide chains and 2 outer D domains. In the final stage of blood coagulation, thrombin cleaves the fibrinopeptides A and B in a sequential manner from the amino termini of the A␣-and B␤-chains. Cleavage occurs between residues R16 (single-letter amino acid abbreviations) and G17 of the A␣-chain and residues R14 and G15 of the B␤-chain, exposing the A and B polymerization sites. Resultant monomers join together to form 2-stranded, halfstaggered protofibrils. It has been shown that protofibrils result from longitudinal D-D interactions and noncovalent contacts between the A and the a sites. The a site is formed by residues 329, 330, 340, and 364 in the carboxy-terminal part of the ␥-chain. 1,2 Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers that increase progressively in thickness and develop branch points. The evolving network is finally stabilized with covalent bonds by the activated factor XIII, resulting in a clot resistant to mechanical disruption.Dysfibrinogenemia is a heritable disorder characterized by structural mutations in any of the 3 polypeptide cha...

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“…Since low-affinity nonsubstrate thrombin binding sites are in the fibrin E domain, it is assumed that a complete absence of low-affinity thrombin binding sites in the BβA68T homozygotes, contributes to reduced thrombin affinity, and then decrease mitigating thrombin procoagulant effects in vivo remain to be fully explored, accordingly a markedly thrombophilia [28]. In BβR14C-and BβR44C-albumin binding families [29], the defective binding of tPA to the fibrin moiety of the abnormal fibrinogen may cause abnormal tPA-mediated plasminogen activation and prolongation of blood clot lysis. An impairment of fibrinolysis in these cases was associated with thrombosis [30].…”

Section: Discussionmentioning

confidence: 99%

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from γAsn319, Asp320 deletion dysfibrinogen, Otsu I

Terasawa

Kani

Hongo

et al. 2006

Thrombosis Research

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Introduction: We have reported a heterozygous dysfibrinogenemia, fibrinogen Otsu I, caused by the deletion of γAsn319 and γAsp320, which was originally identified in the dysfibrinogen Vlissingen/Frankfurt IV (V/FIV) associated with thrombosis. Unlike the V/FIV family, the Otsu propositus showed no thrombotic tendencies. To analyze the relationship between thrombosis and the heterozygous plasma variant fibrinogen, we used purified plasma fibrinogen from the Otsu patient and compared it with a normal control. Materials and Methods:Thrombin-induced fibrin clot formation and clot structure were observed by fibrin polymerization and scanning electron microscopy, respectively. For in vitro observation of fibrinolysis, plasmin generation and clot lysis assays were performed by the addition of tissue type plasminogen activation (tPA) and plasminogen. Results and Conclusions:Polymerization of Otsu was markedly impaired, while fibrin fibers were much thicker and the density of the bundles of fibrin fibers was less and porous compared with normal. Lysis of the Otsu clot was not significantly different from normal when a tPA and plasminogen mixture was overlaid onto the clots. For Otsu, the penetration of the tPA/plasminogen mixture into the clot was much faster than normal and the protection against plasmin cleavage was impaired, however, tPA-induced plasmin activation of the Otsu fibrin was slower than that of normal fibrin, resulting in a clot lysis of Otsu similar to normal.

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Abnormal fibrinogens IJmuiden (B beta Arg14----Cys) and Nijmegen (B beta Arg44----Cys) form disulfide-linked fibrinogen-albumin complexes.

Cited by 59 publications

References 30 publications

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from γAsn319, Asp320 deletion dysfibrinogen, Otsu I

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